Human extracellular signal regulated kinases

ABSTRACT

ERK-7 polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilizing ERK-7 polypeptides and polynucleotides in diagnostic assays.

FIELD OF THE INVENTION

This invention relates to newly identified polypeptides andpolynucleotides encoding such polypeptides sometimes hereinafterreferred to as “novel human extracellular signal regulated kinases(ERK-7)”, to their use in diagnosis and in identifying compounds thatmay be agonists, antagonists that are potentially useful in therapy, andto production of such polypeptides and polynucleotides.

BACKGROUND OF THE INVENTION

The drug discovery process is currently undergoing a fundamentalrevolution as it embraces “functional genomics”, that is, highthroughput genome- or gene-based biology. This approach as a means toidentify genes and gene products as therapeutic targets is rapidlysuperseding earlier approaches based on “positional cloning”. Aphenotype, that is a biological function or genetic disease, would beidentified and this would then be tracked back to the responsible gene,based on its genetic map position.

Functional genomics relies heavily on high-throughput DNA sequencingtechnologies and the various tools of bioinformatics to identify genesequences of potential interest from the many molecular biologydatabases now available. There is a continuing need to identify andcharacterise further genes and their related polypeptides/proteins, astargets for drug discovery.

SUMMARY OF THE INVENTION

The present invention relates to ERK-7, in particular ERK-7 polypeptidesand ERK-7 polynucleotides, recombinant materials and methods for theirproduction. Such polypeptides and polynucleotides are of interest inrelation to methods of treatment of certain diseases, including, but notlimited to, cancers, especially of the breast, ovary, colon, kidney andpancreas and inflammatory diseases hereinafter referred to as “diseasesof the invention”. In a further aspect, the invention relates to methodsfor identifying agonists and antagonists (e.g., inhibitors) using thematerials provided by the invention, and treating conditions associatedwith ERK-7 imbalance with the identified compounds. In a still furtheraspect, the invention relates to diagnostic assays for detectingdiseases associated with inappropriate ERK-7 activity or levels.

DESCRIPTION OF THE INVENTION

In a first aspect, the present invention relates to ERK-7 polypeptides.Such polypeptides include:

-   -   (a) a polypeptide encoded by a polynucleotide comprising the        sequence of SEQ ID NO:1;    -   (b) a polypeptide comprising a polypeptide sequence having at        least 95%, 96%, 97%, 98%, or 99% identity to the polypeptide        sequence of SEQ ID NO:2;    -   (c) a polypeptide comprising the polypeptide sequence of SEQ ID        NO:2;    -   (d) a polypeptide having at least 95%, 96%, 97%, 98%, or 99%        identity to the polypeptide sequence of SEQ ID NO:2;    -   (e) the polypeptide sequence of SEQ ID NO:2; and    -   (f) A polypeptide having or comprising a polypeptide sequence        that has an Identity Index of 0.95, 0.96, 0.97, 0.98, or 0.99        compared to the polypeptide sequence of SEQ ID NO:2;    -   (g) fragments and variants of such polypeptides in (a) to (f).

Polypeptides of the present invention are believed to be members of theextracellular signal regulated kinase family of polypeptides. They aretherefore of interest because This invention describes the seventhmember of the ERK family in humans, revealed in a RDA analysis of larynxtumor tissue. It is homologous to the seventh ERK family member of ratat both the nucleotide and translated protein level. Electronic Northernanalysis of cDNA fragments from the novel ERK-7 show that it is almostexclusively expressed in transformed tissue. The electronic Northernfinds Erk-7 prinarily in breast, ovarian and larynx carcinomas.

Six human extracellular signal regulated kinases (ERKs) have beenidentified. They are involved in the transmission of the extracellularsignals by which growth factor receptors to activate genetranscription—thus, ERK-1 lies in the Ras-MAP Kinase pathway andactivates by phosphorylation transcription factors such as Jun and Fos.

Rat Erk-7, in contrast to Erk-1–6, is not regulated by commonextracellular signals, but by the interactions of the C-terminal proteindomain with intracellular machinery. The amino terminal of the humanhomologue is as yet incomplete—but the existing sequence contains a welldefined C-terminal domain and some 70 amino acids homologous to theC-terminal of the rat Erk-7 kinase domain. The C-terminal domain isthought to be responsible for localization, activation and cell growth(Abe et al., 1999),

The established biological role of Erks, the homology of the discoveryto rat Erk7, and its restricted distribution in cancerous tissuessuggests that Erk-7 is a protein with an important role in cancerdevelopment, and a potential therapeutic target.

The biological properties of the ERK-7 are hereinafter referred to as“biological activity of ERK-7” or “ERK-7 activity”. Preferably, apolypeptide of the present invention exhibits at least one biologicalactivity of ERK-7.

Polypeptides of the present invention also includes variants of theaforementioned polypeptides, including all allelic forms and splicevariants. Such polypeptides vary from the reference polypeptide byinsertions, deletions, and substitutions that may be conservative ornon-conservative, or any combination thereof, Particularly preferredvariants are those in which several, for instance from 50 to 30, from 30to 20, from 20 to 10, from 10 to 5, from 5 to 3, from 3 to 2, from 2 to1 or 1 amino acids are inserted, substituted, or deleted, in anycombination.

Preferred fragments of polypeptides of the present invention include anisolated polypeptide comprising an amino acid sequence having at least30, 50 or 100 contiguous amino acids from the amino acid sequence of SEQID NO: 2, or an isolated polypeptide comprising an amino acid sequencehaving at least 30, 50 or 100 contiguous amino acids truncated ordeleted from the amino acid sequence of SEQ ID NO: 2. Preferredfragments are biologically active fragments that mediate the biologicalactivity of ERK-7, including those with a similar activity or animproved activity, or with a decreased undesirable activity. Alsopreferred are those fragments that are antigenic or immunogenic in ananimal, especially in a human.

Fragments of the polypeptides of the invention may be employed forproducing the corresponding full-length polypeptide by peptidesynthesis; therefore, these variants may be employed as intermediatesfor producing the full-length polypeptides of the invention. Thepolypeptides of the present invention may be in the form of the “mature”protein or may be a part of a larger protein such as a precursor or afusion protein. It is often advantageous to include an additional aminoacid sequence that contains secretory or leader sequences,pro-sequences, sequences that aid in purification, for instance multiplehistidine residues, or an additional sequence for stability duringrecombinant production.

Polypeptides of the present invention can be prepared in any suitablemanner, for instance by isolation form naturally occurring sources, fromgenetically engineered host cells comprising expression systems (videinfra) or by chemical synthesis, using for instance automated peptidesynthesizers, or a combination of such methods. Means for preparing suchpolypeptides are well understood in the art.

In a further aspect, the present invention relates to ERK-7polynucleotides. Such polynucleotides include:

-   -   (a) a polynucleotide comprising a polynucleotide sequence having        at least 95%, 96%, 97%, 98%, or 99% identity to the        polynucleotide sequence of SEQ ID NO:1;    -   (b) a polynucleotide comprising the polynucleotide of SEQ ID        NO:1;    -   (c) a polynucleotide having at least 95%, 96%, 97%, 98%, or 99%        identity to the polynucleotide of SEQ ID NO:1;    -   (d) the isolated polynucleotide of SEQ ID NO:1;    -   (e) a polynucleotide comprising a polynucleotide sequence        encoding a polypeptide sequence having at least 95%, 96%, 97%,        98%, or 99% identity to the polypeptide sequence of SEQ ID NO:2;    -   (f) a polynucleotide comprising a polynucleotide sequence        encoding the polypeptide of SEQ ID NO:2;    -   (g) a polynucleotide having a polynucleotide sequence encoding a        polypeptide sequence having at least 95%, 96%, 97%, 98%, or 99%        identity to the polypeptide sequence of SEQ ID NO:2;    -   (h) a polynucleotide encoding the polypeptide of SEQ ID NO:2;    -   (i) a polynucleotide having or comprising a polynucleotide        sequence that has an Identity Index of 0.95, 0.96, 0.97, 0.98,        or 0.99 compared to the polynucleotide sequence of SEQ ID NO:1;    -   (j) a polynucleotide having or comprising a polynucleotide        sequence encoding a polypeptide sequence that has an Identity        Index of 0.95, 0.96, 0.97, 0.98, or 0.99 compared to the        polypeptide sequence of SEQ ID NO:2; and    -   polynucleotides that are fragments and variants of the above        mentioned polynucleotides or that are complementary to above        mentioned polynucleotides, over the entire length thereof.

Preferred fragments of polynucleotides of the present invention includean isolated polynucleotide comprising an nucleotide sequence having atleast 15, 30, 50 or 100 contiguous nucleotides from the sequence of SEQID NO: 1, or a polynucleotide comprising an sequence having at least 30,50 or 100 contiguous nucleotides truncated or deleted from the sequenceof SEQ ID NO: 1.

Preferred variants of polynucleotides of the present invention includesplice variants, allelic variants, and polymorphisms, includingpolynucleotides having one or more single nucleotide polymorphisms(SNPs).

Polynucleotides of the present invention also include polynucleotidesencoding polypeptide variants that comprise the amino acid sequence ofSEQ ID NO:2 and in which several, for instance from 50 to 30, from 30 to20, from 20 to 10, from 10 to 5, from 5 to 3, from 3 to 2, from 2 to 1or 1 amino acid residues are substituted, deleted or added, in anycombination.

In a further aspect, the present invention provides polynucleotides thatare RNA transcripts of the DNA sequences of the present invention.Accordingly, there is provided an RNA polynucleotide that:

-   -   (a) comprises an RNA transcript of the DNA sequence encoding the        polypeptide of SEQ ID NO:2;    -   (b) is the RNA transcript of the DNA sequence encoding the        polypeptide of SEQ ID NO:2;    -   (c) comprises an RNA transcript of the DNA sequence of SEQ ID        NO:1; or    -   (d) is the RNA transcript of the DNA sequence of SEQ ID NO:1;        and RNA polynucleotides that are complementary thereto.

The polynucleotide sequence of SEQ ID NO:1 shows homology with Abe, M.K., Kuo, W. L., Hershenson, M. B., and Rosner, M. R. (1999).Extracellular signal-regulated kinase 7 (ERK7), a novel ERK with aC-terminal domain that regulates its activity, its cellularlocalization, and cell growth. MOLECULAR.AND.CELLULAR.BIOLOGY. 19,1301–1312.

The polynucleotide sequence of SEQ ID NO:1 is a cDNA sequence thatencodes the polypeptide of SEQ ID NO:2. The polynucleotide sequenceencoding the polypeptide of SEQ ID NO:2 may be identical to thepolypeptide encoding sequence of SEQ ID NO:1 or it may be a sequenceother than SEQ ID NO:1, which, as a result of the redundancy(degeneracy) of the genetic code, also encodes the polypeptide of SEQ IDNO:2. The polypeptide of the SEQ ID NO:2 is related to other proteins ofthe Erk family, having homology and/or structural similarity with RatErk-7 (Abe, M. K., Kuo. W. L., Hershenson, M. B., and Rosner, M. R.(1999). Extracellular signal-regulated kinase 7 (ERK7), a novel ERK witha C-terminal domain that regulates its activity, its cellularlocalization, and cell growth. MOLECULAR.AND.CELLULAR.BIOLOGY. 19,1301–1312).

Preferred polypeptides and polynucleotides of the present invention areexpected to have, inter alia, similar biological functions/properties totheir homologous polypeptides and polynucleotides. Furthermore,preferred polypeptides and polynucleotides of the present invention haveat least one ERK-7 activity.

Polynucleotides of the present invention may be obtained using standardcloning and screening techniques from a cDNA library derived from mRNAin cells of human primary breast cancer, colon adenocarcinoma,peripheral blood neutrophils and ovarian adenocarcinoma, (see forinstance, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2ndEd., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1989)). Polynucleotides of the invention can also be obtained fromnatural sources such as genomic DNA libraries or can be synthesizedusing well known and commercially available techniques.

When polynucleotides of the present invention are used for therecombinant production of polypeptides of the present invention, thepolynucleotide may include the coding sequence for the maturepolypeptide, by itself, or the coding sequence for the maturepolypeptide in reading frame with other coding sequences, such as thoseencoding a leader or secretory sequence, a pre-, or pro- orprepro-protein sequence, or other fusion peptide portions. For example,a marker sequence that facilitates purification of the fused polypeptidecan be encoded. In certain preferred embodiments of this aspect of theinvention, the marker sequence is a hexa-histidine peptide, as providedin the pQE vector (Qiagen, Inc.) and described in Gentz et al., ProcNatl Acad Sci USA (1989) 86:821–824, or is an HA tag. The polynucleotidemay also contain non-coding 5′ and 3′ sequences, such as transcribed,non-translated sequences, splicing and polyadenylation signals, ribosomebinding sites and sequences that stabilize mRNA.

Polynucleotides that are identical, or have sufficient identity to apolynucleotide sequence of SEQ ID NO:1, may be used as hybridizationprobes for cDNA and genomic DNA or as primers for a nucleic acidamplification reaction (for instance, PCR). Such probes and primers maybe used to isolate full-length cDNAs and genomic clones encodingpolypeptides of the present invention and to isolate cDNA and genomicclones of other genes (including genes encoding paralogs from humansources and orthologs and paralogs from species other than human) thathave a high sequence similarity to SEQ ID NO:1, typically at least 95%identity. Preferred probes and primers will generally comprise at least15 nucleotides, preferably, at least 30 nucleotides and may have atleast 50, if not at least 100 nucleotides. Particularly preferred probeswill have between 30 and 50 nucleotides. Particularly preferred primerswill have between 20 and 25 nucleotides.

A polynucleotide encoding a polypeptide of the present invention,including homologs from species other than human, may be obtained by aprocess comprising the steps of screening a library under stringenthybridization conditions with a labeled probe having the sequence of SEQID NO: 1 or a fragment thereof, preferably of at least 15 nucleotides;and isolating full-length cDNA and genomic clones containing saidpolynucleotide sequence. Such hybridization techniques are well known tothe skilled artisan. Preferred stringent hybridization conditionsinclude overnight incubation at 420° C. in a solution comprising: 50%formamide, 5×SSC (150 mM NaCI, 15 mM trisodium citrate), 50 mM sodiumphosphate (pH 7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20microgram/ml denatured, sheared salmon sperm DNA; followed by washingthe filters in 0.1×SSC at about 65° C. Thus the present invention alsoincludes isolated polynucleotides, preferably with a nucleotide sequenceof at least 100, obtained by screening a library under stringenthybridization conditions with a labeled probe having the sequence of SEQID NO:1 or a fragment thereof, preferably of at least 15 nucleotides.

The skilled artisan will appreciate that, in many cases, an isolatedcDNA sequence will be incomplete, in that the region coding for thepolypeptide does not extend all the way through to the 5′ terminus. Thisis a consequence of reverse transcriptase, an enzyme with inherently low“processivity” (a measure of the ability of the enzyme to remainattached to the template during the polymerisation reaction), failing tocomplete a DNA copy of the mRNA template during first strand cDNAsynthesis.

There are several methods available and well known to those skilled inthe art to obtain full-length cDNAs, or extend short cDNAs, for examplethose based on the method of Rapid Amplification of cDNA ends (RACE)(see, for example, Frohman et al., Proc Nat Acad Sci USA 85, 8998–9002,1988). Recent modifications of the technique, exemplified by theMarathon (trade mark) technology (Clontech Laboratories Inc.) forexample, have significantly simplified the search for longer cDNAs. Inthe Marathon (trade mark) technology, cDNAs have been prepared from mRNAextracted from a chosen tissue and an ‘adaptor’ sequence ligated ontoeach end. Nucleic acid amplification (PCR) is then carried out toamplify the “missing” 5′ end of the cDNA using a combination of genespecific and adaptor specific oligonucleotide primers. The PCR reactionis then repeated using ‘nested’ primers, that is, primers designed toanneal within the amplified product (typically an adapter specificprimer that anneals further 3′ in the adaptor sequence and a genespecific primer that anneals further 5′ in the known gene sequence). Theproducts of this reaction can then be analyzed by DNA sequencing and afull-length cDNA constructed either by joining the product directly tothe existing cDNA to give a complete sequence, or carrying out aseparate full-length PCR using the new sequence information for thedesign of the 5′ primer.

Recombinant polypeptides of the present invention may be prepared byprocesses well known in the art from genetically engineered host cellscomprising expression systems. Accordingly, in a further aspect, thepresent invention relates to expression systems comprising apolynucleotide or polynucleotides of the present invention, to hostcells which are genetically engineered with such expression systems andto the production of polypeptides of the invention by recombinanttechniques. Cell-free translation systems can also be employed toproduce such proteins using RNAs derived from the DNA constructs of thepresent invention.

For recombinant production, host cells can be genetically engineered toincorporate expression systems or portions thereof for polynucleotidesof the present invention. Polynucleotides may be introduced into hostcells by methods described in many standard laboratory manuals, such asDavis et al., Basic Methods in Molecular Biology (1986) and Sambrook etal.(ibid). Preferred methods of introducing polynucleotides into hostcells include, for instance, calcium phosphate transfection,DEAE-dextran mediated transfection, transvection, micro-injection,cationic lipid-mediated transfection, electroporation, transduction,scrape loading, ballistic introduction or infection.

Representative examples of appropriate hosts include bacterial cells,such as Streptococci, Staphylococci, E. coli, Streptomyces and Bacillussubtilis cells; fungal cells, such as yeast cells and Aspergillus cells;insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animalcells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanomacells; and plant cells.

A great variety of expression systems can be used, for instance,chromosomal, episomal and virus-derived systems, e.g., vectors derivedfrom bacterial plasmids, from bacteriophage, from transposons, fromyeast episomes from insertion elements, from yeast chromosomal elements,from viruses such as baculoviruses, papova viruses, such as SV40,vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies virusesand retroviruses, and vectors derived from combinations thereof, such asthose derived from plasmid and bacteriophage genetic elements, such ascosmids and phagemids. The expression systems may contain controlregions that regulate as well as engender expression. Generally, anysystem or vector that is able to maintain, propagate or express apolynucleotide to produce a polypeptide in a host may be used. Theappropriate polynucleotide sequence may be inserted into an expressionsystem by any of a variety of well-known and routine techniques, suchas, for example, those set forth in Sambrook et al., (ibid). Appropriatesecretion signals may be incorporated into the desired polypeptide toallow secretion of the translated protein into the lumen of theendoplasmic reticulum, the periplasmic space or the extracellularenvironment. These signals may be endogenous to the polypeptide or theymay be heterologous signals.

If a polypeptide of the present invention is to be expressed for use inscreening assays, it is generally preferred that the polypeptide beproduced at the surface of the cell. In this event, the cells may beharvested prior to use in the screening assay. If the polypeptide issecreted into the medium, the medium can be recovered in order torecover and purify the polypeptide. If produced intracellularly, thecells must first be lysed before the polypeptide is recovered.

Polypeptides of the present invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Most preferably, highperformance liquid chromatography is employed for purification. Wellknown techniques for refolding proteins may be employed to regenerateactive conformation when the polypeptide is denatured duringintracellular synthesis, isolation and/or purification.

Polynucleotides of the present invention may be used as diagnosticreagents, through detecting mutations in the associated gene. Detectionof a mutated form of the gene characterized by the polynucleotide of SEQID NO:1 in the cDNA or genomic sequence and which is associated with adysfunction will provide a diagnostic tool that can add to, or define, adiagnosis of a disease, or susceptibility to a disease, which resultsfrom under-expression, over-expression or altered spatial or temporalexpression of the gene. Individuals carrying mutations in the gene maybe detected at the DNA level by a variety of techniques well known inthe art.

Nucleic acids for diagnosis may be obtained from a subject's cells, suchas from blood, urine, saliva, tissue biopsy or autopsy material. Thegenomic DNA may be used directly for detection or it may be amplifiedenzymatically by using PCR, preferably RT-PCR, or other amplificationtechniques prior to analysis. RNA or cDNA may also be used in similarfashion. Deletions and insertions can be detected by a change in size ofthe amplified product in comparison to the normal genotype. Pointmutations can be identified by hybridizing amplified DNA to labeledERK-7 nucleotide sequences. Perfectly matched sequences can bedistinguished from mismatched duplexes by RNase digestion or bydifferences in melting temperatures. DNA sequence difference may also bedetected by alterations in the electrophoretic mobility of DNA fragmentsin gels, with or without denaturing agents, or by direct DNA sequencing(see, for instance, Myers et al., Science (1985) 230:1242). Sequencechanges at specific locations may also be revealed by nucleaseprotection assays, such as RNase and S1 protection or the chemicalcleavage method (see Cotton et al., Proc Natl Acad Sci USA (1985) 85:4397–4401).

An array of oligonucleotides probes comprising ERK-7 polynucleotidesequence or fragments thereof can be constructed to conduct efficientscreening of e.g., genetic mutations. Such arrays are preferably highdensity arrays or grids. Array technology methods are well known andhave general applicability and can be used to address a variety ofquestions in molecular genetics including gene expression, geneticlinkage, and genetic variability, see, for example, M. Chee et al.,Science, 274, 610–613 (1996) and other references cited therein.

Detection of abnormally decreased or increased levels of polypeptide ormRNA expression may also be used for diagnosing or determiningsusceptibility of a subject to a disease of the invention. Decreased orincreased expression can be measured at the RNA level using any of themethods well known in the art for the quantitation of polynucleotides,such as, for example, nucleic acid amplification, for instance PCR,RT-PCR, RNase protection, Northern blotting and other hybridizationmethods. Assay techniques that can be used to determine levels of aprotein, such as a polypeptide of the present invention, in a samplederived from a host are well-known to those of skill in the art. Suchassay methods include radio-immunoassays, competitive-binding assays,Western Blot analysis and ELISA assays.

Thus in another aspect, the present invention relates to a diagnostickit comprising:

-   -   (a) a polynucleotide of the present invention, preferably the        nucleotide sequence of SEQ ID NO: 1, or a fragment or an RNA        transcript thereof;    -   (b) a nucleotide sequence complementary to that of (a);    -   (c) a polypeptide of the present invention, preferably the        polypeptide of SEQ ID NO:2 or a fragment thereof; or    -   (d) an antibody to a polypeptide of the present invention,        preferably to the polypeptide of SEQ ID NO:2.

It will be appreciated that in any such kit, (a), (b), (c) or (d) maycomprise a substantial component. Such a kit will be of use indiagnosing a disease or susceptibility to a disease, particularlydiseases of the invention, amongst others.

The polynucleotide sequences of the present invention are valuable forchromosome localisation studies. The sequence is specifically targetedto and can hybridize with, a particular location on an individual humanchromosome. The mapping of relevant sequences to chromosomes accordingto the present invention is an important first step in correlating thosesequences with gene associated disease. Once a sequence has been mappedto a precise chromosomal location, the physical position of the sequenceon the chromosome can be correlated with genetic map data. Such data arefound in, for example, V. McKusick, Mendelian Inheritance in Man(available on-line through Johns Hopkins University Welch MedicalLibrary). The relationship between genes and diseases that have beenmapped to the same chromosomal region are then identified throughlinkage analysis (co-inheritance of physically adjacent genes). Precisehuman chromosomal localisations for a genomic sequence (gene fragmentetc.) can be determined using Radiation Hybrid (RH) Mapping (Walter, M.Spillett, D., Thomas, P., Weissenbach, J., and Goodfellow, P., (1994) Amethod for constructing radiation hybrid maps of whole genomes, NatureGenetics 7, 22–28). A number of RH panels are available from ResearchGenetics (Huntsville, Ala., USA) e.g. the GeneBridge4 RH panel (Hum MolGenet 1996 March;5(3):339–46 A radiation hybrid map of the human genome.Gyapay G, Schmitt K, Fizames C, Jones H, Vega-Czarny N, Spillett D,Muselet D, Prud'Homme J F, Dib C, Auffray C, Morissette J, WeissenbachJ, Goodfellow P N). To determine the chromosomal location of a geneusing this panel, 93 PCRs are performed using primers designed from thegene of interest on RH DNAs. Each of these DNAs contains random humangenomic fragments maintained in a hamster background (human/hamsterhybrid cell lines). These PCRs result in 93 scores indicating thepresence or absence of the PCR product of the gene of interest. Thesescores are compared with scores created using PCR products from genomicsequences of known location. This comparison is conducted athttp://www.genome.wi.mit.edu/.

The polynucleotide sequences of the present invention are also valuabletools for tissue expression studies. Such studies allow thedetermination of expression patterns of polynucleotides of the presentinvention which may give an indication as to the expression patterns ofthe encoded polypeptides in tissues, by detecting the mRNAs that encodethem. The techniques used are well known in the art and include in situhydridization techniques to clones arrayed on a grid, such as cDNAmicroarray hybridization (Schena et al, Science, 270, 467–470, 1995 andShalon et al, Genome Res, 6, 639–645, 1996) and nucleotide amplificationtechniques such as PCR. A preferred method uses the TAQMAN (Trade mark)technology available from Perkin Elmer. Results from these studies canprovide an indication of the normal function of the polypeptide in theorganism. In addition, comparative studies of the normal expressionpattern of mRNAs with that of mRNAs encoded by an alternative form ofthe same gene (for example, one having an alteration in polypeptidecoding potential or a regulatory mutation) can provide valuable insightsinto the role of the polypeptides of the present invention, or that ofinappropriate expression thereof in disease. Such inappropriateexpression may be of a temporal, spatial or simply quantitative nature.

A further aspect of the present invention relates to antibodies. Thepolypeptides of the invention or their fragments, or cells expressingthem, can be used as immunogens to produce antibodies that areimmunospecific for polypeptides of the present invention. The term“immunospecific” means that the antibodies have substantially greateraffinity for the polypeptides of the invention than their affinity forother related polypeptides in the prior art.

Antibodies generated against polypeptides of the present invention maybe obtained by administering the polypeptides or epitope-bearingfragments, or cells to an animal, preferably a non-human animal, usingroutine protocols. For preparation of monoclonal antibodies, anytechnique which provides antibodies produced by continuous cell linecultures can be used. Examples include the hybridoma technique (Kohler,G. and Milstein, C., Nature (1975) 256:495–497), the trioma technique,the human B-cell hybridoma technique (Kozbor et al., Immunology Today(1983) 4:72) and the EBV-hybridoma technique (Cole et al., MonoclonalAntibodies and Cancer Therapy, 77–96, Alan R. Liss, Inc., 1985).

Techniques for the production of single chain antibodies, such as thosedescribed in U.S. Pat. No. 4,946,778, can also be adapted to producesingle chain antibodies to polypeptides of this invention. Also,transgenic mice, or other organisms, including other mammals, may beused to express humanized antibodies.

The above-described antibodies may be employed to isolate or to identifyclones expressing the polypeptide or to purify the polypeptides byaffinity chromatography. Antibodies against polypeptides of the presentinvention may also be employed to treat diseases of the invention,amongst others.

Polypeptides and polynucleotides of the present invention may also beused as vaccines. Accordingly, in a further aspect, the presentinvention relates to a method for inducing an immunological response ina mammal that comprises inoculating the mammal with a polypeptide of thepresent invention, adequate to produce antibody and/or T cell immuneresponse, including, for example, cytokine-producing T cells orcytotoxic T cells, to protect said animal from disease, whether thatdisease is already established within the individual or not. Animmunological response in a mammal may also be induced by a methodcomprises delivering a polypeptide of the present invention via a vectordirecting expression of the polynucleotide and coding for thepolypeptide in vivo in order to induce such an immunological response toproduce antibody to protect said animal from diseases of the invention.One way of administering the vector is by accelerating it into thedesired cells as a coating on particles or otherwise. Such nucleic acidvector may comprise DNA, RNA, a modified nucleic acid, or a DNA/RNAhybrid. For use a vaccine, a polypeptide or a nucleic acid vector willbe normally provided as a vaccine formulation (composition). Theformulation may further comprise a suitable carrier. Since a polypeptidemay be broken down in the stomach, it is preferably administeredparenterally (for instance, subcutaneous, intra-muscular, intravenous,or intra-dermal injection). Formulations suitable for parenteraladministration include aqueous and non-aqueous sterile injectionsolutions that may contain anti-oxidants, buffers, bacteriostats andsolutes that render the formulation instonic with the blood of therecipient; and aqueous and non-aqueous sterile suspensions that mayinclude suspending agents or thickening agents. The formulations may bepresented in unit-dose or multi-dose containers, for example, sealedampoules and vials and may be stored in a freeze-dried conditionrequiring only the addition of the sterile liquid carrier immediatelyprior to use. The vaccine formulation may also include adjuvant systemsfor enhancing the immunogenicity of the formulation, such as oil-inwater systems and other systems known in the art. The dosage will dependon the specific activity of the vaccine and can be readily determined byroutine experimentation.

Polypeptides of the present invention have one or more biologicalfunctions that are of relevance in one or more disease states, inparticular the diseases of the invention hereinbefore mentioned. It istherefore useful to identify compounds that stimulate or inhibit thefunction or level of the polypeptide. Accordingly, in a further aspect,the present invention provides for a method of screening compounds toidentify those that stimulate or inhibit the function or level of thepolypeptide. Such methods identify agonists or antagonists that may beemployed for therapeutic and prophylactic purposes for such diseases ofthe invention as hereinbefore mentioned. Compounds may be identifiedfrom a variety of sources, for example, cells, cell-free preparations,chemical libraries, collections of chemical compounds, and naturalproduct mixtures. Such agonists or antagonists so-identified may benatural or modified substrates, ligands, receptors, enzymes, etc., asthe case may be, of the polypeptide; a structural or functional mimeticthereof (see Coligan et al., Current Protocols in Immunology1(2):Chapter 5 (1991)) or a small molecule. Such small moleculespreferably have a molecular weight below 2,000 daltons, more preferablybetween 300 and 1,000 daltons, and most preferably between 400 and 700daltons. It is preferred that these small molecules are organicmolecules.

The screening method may simply measure the binding of a candidatecompound to the polypeptide, or to cells or membranes bearing thepolypeptide, or a fusion protein thereof, by means of a label directlyor indirectly associated with the candidate compound. Alternatively, thescreening method may involve measuring or detecting (qualitatively orquantitatively) the competitive binding of a candidate compound to thepolypeptide against a labeled competitor (e.g. agonist or antagonist).Further, these screening methods may test whether the candidate compoundresults in a signal generated by activation or inhibition of thepolypeptide, using detection systems appropriate to the cells bearingthe polypeptide. Inhibitors of activation are generally assayed in thepresence of a known agonist and the effect on activation by the agonistby the presence of the candidate compound is observed. Further, thescreening methods may simply comprise the steps of mixing a candidatecompound with a solution containing a polypeptide of the presentinvention, to form a mixture, measuring a ERK-7 activity in the mixture,and comparing the ERK-7 activity of the mixture to a control mixturewhich contains no candidate compound.

Polypeptides of the present invention may be employed in conventionallow capacity screening methods and also in high-throughput screening(HTS) formats. Such HTS formats include not only the well-establisheduse of 96- and, more recently, 384-well micotiter plates but alsoemerging methods such as the nanowell method described by Schullek etal, Anal Biochem., 246, 20–29, (1997).

Fusion proteins, such as those made from Fc portion and ERK-7polypeptide, as hereinbefore described, can also be used forhigh-throughput screening assays to identify antagonists for thepolypeptide of the present invention (see D. Bennett et al., J MolRecognition, 8:52–58 (1995); and K. Johanson et al., J Biol Chem,270(16):9459–9471 (1995)).

Screening Techniques

The polynucleotides, polypeptides and antibodies to the polypeptide ofthe present invention may also be used to configure screening methodsfor detecting the effect of added compounds on the production of mRNAand polypeptide in cells. For example, an ELISA assay may be constructedfor measuring secreted or cell associated levels of polypeptide usingmonoclonal and polyclonal antibodies by standard methods known in theart. This can be used to discover agents that may inhibit or enhance theproduction of polypeptide (also called antagonist or agonist,respectively) from suitably manipulated cells or tissues.

A polypeptide of the present invention may be used to identify membranebound or soluble receptors, if any, through standard receptor bindingtechniques known in the art. These include, but are not limited to,ligand binding and crosslinking assays in which the polypeptide islabeled with a radioactive isotope (for instance, ¹²⁵¹I), chemicallymodified (for instance, biotinylated), or fused to a peptide sequencesuitable for detection or purification, and incubated with a source ofthe putative receptor (cells, cell membranes, cell supernatants, tissueextracts, bodily fluids). Other methods include biophysical techniquessuch as surface plasmon resonance and spectroscopy. These screeningmethods may also be used to identify agonists and antagonists of thepolypeptide that compete with the binding of the polypeptide to itsreceptors, if any. Standard methods for conducting such assays are wellunderstood in the art.

Examples of antagonists of polypeptides of the present invention includeantibodies or, in some cases, oligonucleotides or proteins that areclosely related to the ligands, substrates, receptors, enzymes, etc., asthe case may be, of the polypeptide, e.g., a fragment of the ligands,substrates, receptors, enzymes, etc.; or a small molecule that bind tothe polypeptide of the present invention but do not elicit a response,so that the activity of the polypeptide is prevented.

Screening methods may also involve the use of transgenic technology andERK-7 gene. The art of constructing transgenic animals is wellestablished. For example, the ERK-7 gene may be introduced throughmicroinjection into the male pronucleus of fertilized oocytes,retroviral transfer into pre- or post-implantation embryos, or injectionof genetically modified, such as by electroporation, embryonic stemcells into host blastocysts. Particularly useful transgenic animals areso-called “knock-in” animals in which an animal gene is replaced by thehuman equivalent within the genome of that animal. Knock-in transgenicanimals are useful in the drug discovery process, for target validation,where the compound is specific for the human target. Other usefultransgenic animals are so-called “knock-out” animals in which theexpression of the animal ortholog of a polypeptide of the presentinvention and encoded by an endogenous DNA sequence in a cell ispartially or completely annulled. The gene knock-out may be targeted tospecific cells or tissues, may occur only in certain cells or tissues asa consequence of the limitations of the technology, or may occur in all,or substantially all, cells in the animal. Transgenic animal technologyalso offers a whole animal expression-cloning system in which introducedgenes are expressed to give large amounts of polypeptides of the presentinvention

Screening kits for use in the above described methods form a furtheraspect of the present invention. Such screening kits comprise:

-   -   (a) a polypeptide of the present invention;    -   (b) a recombinant cell expressing a polypeptide of the present        invention;    -   (c) a cell membrane expressing a polypeptide of the present        invention; or    -   (d) an antibody to a polypeptide of the present invention:    -   which polypeptide is preferably that of SEQ ID NO:2.

It will be appreciated that in any such kit, (a), (b), (c) or (d) maycomprise a substantial component.

Glossary

The following definitions are provided to facilitate understanding ofcertain terms used frequently hereinbefore. “Antibodies” as used hereinincludes polyclonal and monoclonal antibodies, chimeric, single chain,and humanized antibodies, as well as Fab fragments, including theproducts of an

Fab or other immunoglobulin expression library.

“Isolated” means altered “by the hand of man” from its natural state,i.e., if it occurs in nature, it has been changed or removed from itsoriginal environment, or both. For example, a polynucleotide or apolypeptide naturally present in a living organism is not “isolated,”but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is “isolated”, as the term is employedherein. Moreover, a polynucleotide or polypeptide that is introducedinto an organism by transformation, genetic manipulation or by any otherrecombinant method is “isolated” even if it is still present in saidorganism, which organism may be living or non-living.

“Polynucleotide” generally refers to any polyribonucleotide (RNA) orpolydeoxribonucleotide (DNA), which may be unmodified or modified RNA orDNA. “Polynucleotides” include, without limitation, single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions. single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions. Inaddition, “polynucleotide” refers to triple-stranded regions comprisingRNA or DNA or both RNA and DNA. The term “polynucleotide” also includesDNAs or RNAs containing one or more modified bases and DNAs or RNAs withbackbones modified for stability or for other reasons. “Modified” basesinclude, for example, tritylated bases and unusual bases such asinosine. A variety of modifications may be made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically or metabolicallymodified forms of polynucleotides as typically found in nature, as wellas the chemical forms of DNA and RNA characteristic of viruses andcells. “Polynucleotide” also embraces relatively short polynucleotides,often referred to as oligonucleotides.

“Polypeptide” refers to any polypeptide comprising two or more aminoacids joined to each other by peptide bonds or modified peptide bonds,i.e., peptide isosteres. “Polypeptide” refers to both short chains,commonly referred to as peptides, oligopeptides or oligomers, and tolonger chains, generally referred to as proteins. Polypeptides maycontain amino acids other than the 20 gene-encoded amino acids.“Polypeptides” include amino acid sequences modified either by naturalprocesses, such as post-translational processing, or by chemicalmodification techniques that are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications may occur anywhere in a polypeptide, including the peptidebackbone the amino acid side-chains and the amino or carboxyl termini.It will be appreciated that the same type of modification may be presentto the same or varying degrees at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Polypeptides may be branched as a result of ubiquitination, and they maybe cyclic, with or without branching. Cyclic, branched and branchedcyclic polypeptides may result from post-translation natural processesor may be made by synthetic methods. Modifications include acetylation,acylation, ADP-ribosylation, amidation, biotinylation, covalentattachment of flavin, covalent attachment of a heme moiety, covalentattachment of a nucleotide or nucleotide derivative, covalent attachmentof a lipid or lipid derivative, covalent attachment ofphosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cystine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, proteolyticprocessing, phosphorylation, prenylation, racemization, selenoylation,sulfation, transfer-RNA mediated addition of amino acids to proteinssuch as arginylation, and ubiquitination (see, for instance, ProteinsStructure and Molecular Properties, 2nd Ed., T. E. Creighton, W. H.Freeman and Company, New York, 1993; Wold, F., Post-translationalProtein Modifications: Perspectives and Prospects, 1–12, inPost-translational Covalent Modification of Proteins, B. C. Johnson,Ed., Academic Press. New York, 1983; Seifter et al., “Analysis forprotein modifications and nonprotein cofactors”, Meth Enzymol, 182,626–646, 1990, and Rattan et al., “Protein Synthesis: Post-translationalModifications and Aging”, Ann NY Acad Sci, 663, 48–62, 1992).

“Fragment” of a polypeptide sequence refers to a polypeptide sequencethat is shorter than the reference sequence but that retains essentiallythe same biological function or activity as the reference polypeptide.“Fragment” of a polynucleotide sequence refers to a polynucleotidesequence that is shorter than the reference sequence of SEQ ID NO:1.

“Variant” refers to a polynucleotide or polypeptide that differs from areference polynucleotide or polypeptide, but retains the essentialproperties thereof. A typical variant of a polynucleotide differs innucleotide sequence from the reference polynucleotide. Changes in thenucleotide sequence of the variant may or may not alter the amino acidsequence of a polypeptide encoded by the reference polynucleotide.Nucleotide changes may result in amino acid substitutions, additions,deletions, fusions and truncations in the polypeptide encoded by thereference sequence, as discussed below. A typical variant of apolypeptide differs in amino acid sequence from the referencepolypeptide. Generally, alterations are limited so that the sequences ofthe reference polypeptide and the variant are closely similar overalland, in many regions, identical. A variant and reference polypeptide maydiffer in amino acid sequence by one or more substitutions, insertions,deletions in any combination. A substituted or inserted amino acidresidue may or may not be one encoded by the genetic code. Typicalconservative substitutions include Gly, Ala; Val, lle, Leu; Asp, Glu;Asn, Gln; Ser, Thr: Lys, Arg; and Phe and Tyr. A variant of apolynucleotide or polypeptide may be naturally occurring such as anallele, or it may be a variant that is not known to occur naturally.Non-naturally occurring variants of polynucleotides and polypeptides maybe made by mutagenesis techniques or by direct synthesis. Also includedas variants are polypeptides having one or more post-translationalmodifications, for instance glycosylation, phosphorylation, methylation,ADP ribosylation and the like. Embodiments include methylation of theN-terminal amino acid, phosphorylations of serines and threonines andmodification of C-terminal glycines.

“Allele” refers to one of two or more alternative forms of a geneoccurring at a given locus in the genome.

“Polymorphism” refers to a variation in nucleotide sequence (and encodedpolypeptide sequence, if relevant) at a given position in the genomewithin a population.

“Single Nucleotide Polymorphism” (SNP) refers to the occurrence ofnucleotide variability at a single nucleotide position in the genome,within a population. An SNP may occur within a gene or within intergenicregions of the genome. SNPs can be assayed using Allele SpecificAmplification (ASA). For the process at least 3 primers are required. Acommon primer is used in reverse complement to the polymorphism beingassayed. This common primer can be between 50 and 1500 bps from thepolymorphic base. The other two (or more) primers are identical to eachother except that the final 3′ base wobbles to match one of the two (ormore) alleles that make up the polymorphism. Two (or more) PCR reactionsare then conducted on sample DNA, each using the common primer and oneof the Allele Specific Primers.

“Splice Variant” as used herein refers to cDNA molecules produced fromRNA molecules initially transcribed from the same genomic DNA sequencebut which have undergone alternative RNA splicing. Alternative RNAsplicing occurs when a primary RNA transcript undergoes splicing,generally for the removal of introns, which results in the production ofmore than one mRNA molecule each of that may encode different amino acidsequences. The term splice variant also refers to the proteins encodedby the above cDNA molecules.

“Identity” reflects a relationship between two or more polypeptidesequences or two or more polynucleotide sequences, determined bycomparing the sequences. In general, identity refers to an exactnucleotide to nucleotide or amino acid to amino acid correspondence ofthe two polynucleotide or two polypeptide sequences, respectively, overthe length of the sequences being compared.

“% Identity”—For sequences where there is not an exact correspondence, a“% identity” may be determined. In general, the two sequences to becompared are aligned to give a maximum correlation between thesequences. This may include inserting “gaps” in either one or bothsequences, to enhance the degree of alignment. A % identity may bedetermined over the whole length of each of the sequences being compared(so-called global alignment), that is particularly suitable forsequences of the same or very similar length, or over shorter, definedlengths (so-called local alignment), that is more suitable for sequencesof unequal length.

“Similarity” is a further, more sophisticated measure of therelationship between two polypeptide sequences. In general, “similarity”means a comparison between the amino acids of two polypeptide chains, ona residue by residue basis, taking into account not only exactcorrespondences between a between pairs of residues, one from each ofthe sequences being compared (as for identity) but also, where there isnot an exact correspondence, whether, on an evolutionary basis, oneresidue is a likely substitute for the other. This likelihood has anassociated “score” from which the “% similarity” of the two sequencescan then be determined.

Methods for comparing the identity and similarity of two or moresequences are well known in the art. Thus for instance, programsavailable in the Wisconsin Sequence Analysis Package, version 9.1(Devereux J et al, Nucleic Acids Res, 12, 387–395, 1984, available fromGenetics Computer Group, Madison, Wis., USA), for example the programsBESTFIT and GAP, may be used to determine the % identity between twopolynucleotides and the % identity and the % similarity between twopolypeptide sequences. BESTFIT uses the “local homology” algorithm ofSmith and Waterman (J Mol Biol, 147,195–197, 1981, Advances in AppliedMathematics, 2, 482–489, 1981) and finds the best single region ofsimilarity between two sequences. BESTFIT is more suited to comparingtwo polynucleotide or two polypeptide sequences that are dissimilar inlength, the program assuming that the shorter sequence represents aportion of the longer. In comparison, GAP aligns two sequences, findinga “maximum similarity”, according to the algorithm of Neddleman andWunsch (J Mol Biol, 48, 443–453, 1970). GAP is more suited to comparingsequences that are approximately the same length and an alignment isexpected over the entire length. Preferably, the parameters “Gap Weight”and “Length Weight” used in each program are 50 and 3, forpolynucleotide sequences and 12 and 4 for polypeptide sequences,respectively. Preferably, % identities and similarities are determinedwhen the two sequences being compared are optimally aligned.

Other programs for determining identity and/or similarity betweensequences are also known in the art, for instance the BLAST family ofprograms (Altschul S F et al, J Mol Biol, 215, 403–410, 1990, Altschul SF et al, Nucleic Acids Res., 25:389–3402, 1997, available from theNational Center for Biotechnology Information (NCBI), Bethesda, Md., USAand accessible through the home page of the NCBI atwww.ncbi.nim.nih.gov) and FASTA (Pearson W R, Methods in Enzymology,183, 63–99, 1990; Pearson W R and Lipman D J, Proc Nat Acad Sci USA, 85,2444–2448,1988, available as part of the Wisconsin Sequence AnalysisPackage).

Preferably, the BLOSUM62 amino acid substitution matrix (Henikoff S andHenikoff J G, Proc. Nat. Acad Sci. USA, 89, 10915–10919, 1992) is usedin polypeptide sequence comparisons including where nucleotide sequencesare first translated into amino acid sequences before comparison.

Preferably, the program BESTFIT is used to determine the % identity of aquery polynucleotide or a polypeptide sequence with respect to areference polynucleotide or a polypeptide sequence, the query and thereference sequence being optimally aligned and the parameters of theprogram set at the default value, as hereinbefore described.

“Identity Index” is a measure of sequence relatedness which may be usedto compare a candidate sequence (polynucleotide or polypeptide) and areference sequence. Thus, for instance, a candidate polynucleotidesequence having, for example, an Identity Index of 0.95 compared to areference polynucleotide sequence is identical to the reference sequenceexcept that the candidate polynucleotide sequence may include on averageup to five differences per each 100 nucleotides of the referencesequence. Such differences are selected from the group consisting of atleast one nucleotide deletion, substitution, including transition andtransversion, or insertion. These differences may occur at the 5′ or 3′terminal positions of the reference polynucleotide sequence or anywherebetween these terminal positions, interspersed either individually amongthe nucleotides in the reference sequence or in one or more contiguousgroups within the reference sequence. In other words, to obtain apolynucleotide sequence having an Identity Index of 0.95 compared to areference polynucleotide sequence, an average of up to 5 in every 100 ofthe nucleotides of the in the reference sequence may be deleted,substituted or inserted, or any combination thereof, as hereinbeforedescribed. The same applies mutatis mutandis for other values of theIdentity Index, for instance 0.96, 0.97, 0.98 and 0.99.

Similarly, for a polypeptide, a candidate polypeptide sequence having,for example, an Identity Index of 0.95 compared to a referencepolypeptide sequence is identical to the reference sequence except thatthe polypeptide sequence may include an average of up to fivedifferences per each 100 amino acids of the reference sequence. Suchdifferences are selected from the group consisting of at least one aminoacid deletion, substitution, including conservative and non-conservativesubstitution, or insertion. These differences may occur at the amino- orcarboxy-terminal positions of the reference polypeptide sequence oranywhere between these terminal positions, interspersed eitherindividually among the amino acids in the reference sequence or in oneor more contiguous groups within the reference sequence. In other words,to obtain a polypeptide sequence having an Identity Index of 0.95compared to a reference polypeptide sequence, an average of up to 5 inevery 100 of the amino acids in the reference sequence may be deleted,substituted or inserted, or any combination thereof, as hereinbeforedescribed. The same applies mutatis mutandis for other values of theIdentity Index, for instance 0.96, 0.97, 0.98 and 0.99.

The relationship between the number of nucleotide or amino aciddifferences and the Identity Index may be expressed in the followingequation:n _(a) ≦x _(a)−(x _(a) ·I),in which:

-   -   n_(a) is the number of nucleotide or amino acid differences,    -   x_(a) is the total number of nucleotides or amino acids in SEQ        ID NO:1 or SEQ ID NO:2, respectively,    -   I is the Identity Index,    -   · is the symbol for the multiplication operator, and    -   in which any non-integer product of x_(a) and I is rounded down        to the nearest integer prior to subtracting it from x_(a).

“Homolog” is a generic term used in the art to indicate a polynucleotideor polypeptide sequence possessing a high degree of sequence relatednessto a reference sequence. Such relatedness may be quantified bydetermining the degree of identity and/or similarity between the twosequences as hereinbefore defined. Falling within this generic term arethe terms “ortholog”, and “paralog”. “Ortholog” refers to apolynucleotide or polypeptide that is the functional equivalent of thepolynucleotide or polypeptide in another species. “Paralog” refers to apolynucleotideor polypeptide that within the same species which isfunctionally similar.

“Fusion protein” refers to a protein encoded by two, unrelated, fusedgenes or fragments thereof. Examples have been disclosed in U.S. Pat.No. 5541087, 5726044. In the case of Fc-ERK-7, employing animmunoglobulin Fc region as a part of a fusion protein is advantageousfor performing the functional expression of Fc- ERK-7 or fragments ofERK-7, to improve pharmacokinetic properties of such a fusion proteinwhen used for therapy and to generate a dimeric ERK-7. The Fc- ERK-7 DNAconstruct comprises in 5′ to 3′ direction, a secretion cassette, i.e. asignal sequence that triggers export from a mammalian cell, DNA encodingan immunoglobulin Fc region fragment, as a fusion partner, and a DNAencoding ERK-7 or fragments thereof. In some uses it would be desirableto be able to alter the intrinsic functional properties (complementbinding, Fc-Receptor binding) by mutating the functional Fc sides whileleaving the rest of the fusion protein untouched or delete the Fc partcompletely after expression.

All publications and references, including but not limited to patentsand patent applications, cited in this specification are hereinincorporated by reference in their entirety as if each individualpublication or reference were specifically and individually indicated tobe incorporated by reference herein as being fully set forth. Any patentapplication to which this application claims priority is alsoincorporated by reference herein in its entirety in the manner describedabove for publications and references.

1. An isolated polypeptide selected from the group consisting of: (a) apolypeptide encoded by a polynucleotide comprising the sequence of SEQID NO:1; (b) a polypeptide comprising a polypeptide sequence having atleast 95% identity over its entire length to the polypeptide sequence ofSEQ ID NO:2 and which is encoded by a polynucleotide which hybridizes tothe complement of SEQ ID NO:1 under stringent hybridization conditionscomprising overnight incubation at 42° C. in a solution comprising: 50%formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodiumphosphate (pH 7.6), 5× Denhardt's solution, 10% dextran sulphate, and 20microgram/ml denatured, sheared salmon sperm DNA; followed by washingthe filters in 0.1×SSC at 65° C.; (c) a polypeptide having at least 95%identity over its entire length to the polypeptide sequence of SEQ IDNO:2 and which is encoded by a polynucleotide which hybridizes to thecomplement of SEQ ID NO:1 under stringent hybridization conditionscomprising overnight incubation at 42° C. in a solution comprising: 50%formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodiumphosphate (pH 7.6), 5× Denhardt's solution, 10% dextran sulphate, and 20microgram/ml denatured, sheared salmon sperm DNA; followed by washingthe filters in 0.1×SSC at 65° C.; and (d) the polypeptide sequence ofSEQ ID NO:2, wherein said polypeptide of a)–d) has extracelularsignal-regulated kinase (ERK) activity.
 2. The isolated polypeptide ofclaim 1 comprising the polypeptide sequence of SEQ ID NO:2.
 3. Theisolated polypeptide of claim 1 which consists of the polypeptidesequence of SEQ ID NO:2.
 4. A fusion protein comprising anImmunoglobulin Fc-region and a polypeptide of claim
 1. 5. An isolatedpolypeptide of claim 1, which is a polypeptide comprising a polypeptidesequence having at least 95% identity over its entire length to thepolypeptide sequence of SEQ ID NO:2 and which is encoded by apolynucleotide which hybridizes to the complement of SEQ ID NO:1 understringent hybridization conditions comprising overnight incubation at42° C. in a solution comprising: 50% formamide, 5×SSC (150 mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt'ssolution, 10% dextran sulphate, and 20 microgram/ml denatured, shearedsalmon sperm DNA; followed by washing the filters in 0.1×SSC at 65° C.,wherein said polypeptide has extracellular signal-regulated kinase (ERK)activity.
 6. An isolated polypeptide of claim 1, which consists of apolypeptide having at least 95% identity over its entire length to thepolypeptide sequence of SEQ ID NO:2 and which is encoded by apolynucleotide which hybridizes to the complement of SEQ ID NO:1 understringent hybridization conditions comprising overnight incubation at42° C. in a solution comprising: 50% formamide, 5×SSC (150 mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt'ssolution, 10% dextran sulphate, and 20 microgram/ml denatured, shearedsalmon sperm DNA; followed by washing the filters in 0.1×SSC at 65° C.,wherein said polypeptide has extracellular signal-regulated kinase (ERK)activity.
 7. An isolated polypeptide which comprises a polypeptidefragment of SEQ ID NO:2, wherein said polypeptide fragment comprises atleast 100 amino acids and has extracellular signal-regulated kinase(ERK) activity.